High-throughput Nucleotide Sequencing Research Articles

Clinical features of Streptococcus intermedius infection in children: a case series study.

Streptococcus intermedius is an opportunistic pathogen associated with prolonged hospital stays and high mortality rates in adults. However, little is currently known about the clinical features of Streptococcus intermedius infection in children. This retrospective case series study included 40 children diagnosed with Streptococcus intermedius, confirmed through bacterial cultures or high-throughput sequencing. Antibiotic resistance was assessed through susceptibility testing. The site and clinical manifestations were evaluated for all patients. The common infection sites were the abdominal cavity, skin and soft tissue, intracranial, and invasive pulmonary, with the abdominal cavity being the most frequently affected. The drug susceptibility test showed 100% sensitivity to ceftriaxone, levofloxacin, chloramphenicol, vancomycin, and linezolid, 92.6% sensitivity to penicillin, 73.3% resistance to erythromycin, and 76.7% resistance to clindamycin. Besides antibiotic therapy, surgical intervention or pus drainage was often necessary. Lung imaging of four patients revealed pulmonary abscesses, nodules, or encapsulated pleura. Two cases yielded positive culture results, while three were identified as positive through high-throughput nucleotide sequencing of pleural effusion. In children with Streptococcus intermedius infection, emphasis should be placed on the risk of pus or abscess formation. In cases of pulmonary abscess and pleural effusion, especially in male children, Streptococcus intermedius should be suspected even if the culture is negative. Improvements in high-throughput nucleotide sequencing are required to reduce misdiagnosis rates.

Sequencing for novel mutation screening in juvenile polyposis syndrome

Juvenile polyposis syndrome (JPS), a rare polyposis syndrome in childhood, is characterized by multiple diffuse hamartomatous polyps in the gastrointestinal tract (GI) with an estimated incidence of one in 50,000 to 100,000 live births. Although a juvenile polyp is a non-cancerous lesion, affected individuals have an increased risk of GI malignancy. JPS is a disease related to loss-of-function mutations in BMPR1A and SMAD4 involved in the biological maintenance of cellular growth. Pathogenic mutations in those genes have been identified in approximately 50–60% of JPS cases. Our report describes two JPS cases with loss-of-function mutations of BMPR1A identified by a high-throughput nucleotide sequencing. Both cases had a similar clinical manifestation, symptomatic lower GI bleeding. Both patients underwent a total colectomy after histological confirmation. Consequently, Family members who had had genetic confirmation of these mutations were invited to be screened by enteroscopy. JPS is a hereditary disease caused by dominantly inherited loss-of-function of the BMPR1A and SMAD4 genes . Identifying loss-of-function mutations in particular genes allows early screening, diagnosis, and prevention of malignancy among the index cases and their families. High-throughput genome sequencing is a current molecular approach for improving the precision of mutation detection and minimizing time to diagnosis.

Intestinal microbiome and its relationship with necrotizing enterocolitis in very low birth weight preterm infants

Objective: To explore the composition of intestinal microflora prior to onset of necrotizing enterocolitis (NEC) in very low birth weight preterm infants. Methods: This was a multicenter prospective nested case-control study. A total of 46 very low birth weight preterm infants (birth weight <1 500 g and gestional age <35 weeks) within 24 h of life admitted into Neonatal Intensive Care Unit of Children's Hospital of Soochow University and Suzhou Municipal Hospital from April 20 to November 20, 2018 were enrolled. Baseline clinical data and fecal samples of these infants were collected. The subsequent sampling time points were 1st, 4th and 7th day in the first week of life then once per week consecutively. The endpoint of sampling was NEC occurrence, patient discharge or the 8th week post-discharge, whichever came first. Fecal samples were analyzed by 16 S rDNA high-throughput nucleotide sequencing. The control cases were infants without NEC who were matched to the NEC cases with a ratio of 1∶1. The operational taxonomic units (OTU), sequence number and shannon diversity index of the fecal samples were analyzed. Continuous variables were compared with t-test or non-parametric test, and χ2 test or Fisher's exact test was used for categorical variables. Results: There were 23 patients in each group. The gestational age was (29.4±1.8) weeks in NEC group and (29.9±1.6) weeks in control group, including 13 males (57%) and 11 males (48%) in each group, respectively. Species abundance showed that the Firmicutes in both groups decreased temporarily at 7 days of age and then increased with age in control group, but not in NEC group, the Proteobacteria in both groups increased at 7 days of age and then decreased in control group, but kept increasing in NEC group. Regarding the other levels of taxonomy, compared with that of the control group, the NEC group had lower abundance of Proteobacteria, γ-proteobacteria and Enterobacteriaceae at 7 days of age, while higer abundance of Faecalibacterium at 14 days of age, meanwhile, lower Clostridium and Streptococcus at 21 days of age, lower Firmicutes, Clostridia and Clostridium perfringens and higher Proteobacteria and γ-proteobacteria at 28 days of age, these differences were all statistically significant (U=43.00, 43.00, 45.00, 80.00, 74.00, 76.00, 19.00, 8.00, 36.00, 25.00, 25.00,all P<0.05). The shannon index of NEC group was both lower than that of the controls at 21 days of age (2.4 (1.4, 3.0) vs. 3.1 (2.6, 4.0), U=67.00, P=0.027) and 28 days of age (2.4 (1.4, 2.8) vs. 3.9 (3.3, 4.2), U=12.00, P=0.001). Conclusions: The intestinal microflora profile of very low birth weight preterm infants has already changed prior to NEC development. The emergence of differential flora and the reduction of microflora diversity may facilitate early identification and prevention of NEC.

MODY5 Hepatocyte Nuclear Factor 1ß (HNF1ß)-Associated Nephropathy: experience from a regional monogenic diabetes referral centre in Singapore.

From our monogenic diabetes registry set-up at a secondary-care diabetes center, we identified a nontrivial subpopulation (~15%) of maturity-onset diabetes of the young (MODY) among people with young-onset diabetes. In this report, we describe the diagnostic caveats, clinical features and long-term renal-trajectory of people with HNF1B mutations (HNF1B-MODY). Between 2013 and 2020, we received 267 referrals to evaluate MODY from endocrinologists in both public and private practice. Every participant was subjected to a previously reported structured evaluation process, high-throughput nucleotide sequencing and gene-dosage analysis. Out of 40 individuals with confirmed MODY, 4 (10%) had HNF1B-MODY (harboring either a HNF1B whole-gene deletion or duplication). Postsequencing follow-up biochemical and radiological evaluations revealed the known HNF1B-MODY associated systemic-features, such as transaminitis and structural renal-lesions. These anomalies could have been missed without prior knowledge of the nucleotide-sequencing results. Interestingly, preliminary longitudinal observation (up to 15 years) suggested possibly 2 distinct patterns of renal-deterioration (albuminuric vs. nonalbuminuric chronic kidney disease). Monogenic diabetes like HNF1B-MODY may be missed among young-onset diabetes in a resource-limited routine-care clinic. Collaboration with a MODY-evaluation center may fill the care-gap. The long-term renal-trajectories of HNF1B-MODY will require further studies by dedicated registries and international consortium.

Workflow to Mine Frequent DNA Co-methylation Clusters in DNA Methylome Data.

The advances in high-throughput nucleotide sequencing technology revolutionized biomedical research. Vast amount of genomic data rapidly accumulates in a daily basis, which in turn calls for the development of powerful bioinformatics tools and efficient workflows to analyze them. One of the approaches to address the "big data" issue is to mine highly correlated clusters/networks of biological molecules, which may provide rich yet hidden information about the underlying functional, regulatory, or structural relationships among genes, proteins, genomic loci or various types of biological molecules or events. A network mining algorithm lmQCM has recently been developed, which can be applied to mine tightly connected correlation clusters (networks) in large biological data with big sample size, and it guarantees a lower bound of the cluster density. This algorithm has been used in a variety of cancer transcriptomic data to mine gene co-expression networks (GCNs), but it can be applied to any correlational matrix. lmQCM is available through R package lmQCM as well as the online tool package TSUNAMI ( https://biolearns.medicine.iu.edu ).In this study, the purpose is to establish an analytical workflow to apply lmQCM for frequent (consensus)cluster mining in multiple DNA methylation datasets in different cancers and extract the underlying common co-methylation networks for genes.Specifically, the workflow is applied to analyze DNA methylome data across different cancer types using lmQCM. It mines frequent DNA methylation clusters based on individual clustering mining results, identifying common as well as distinctive DNA methylation patterns among different cancer types. This workflow has successfully identified frequent GCNs in 33 types of cancers, thus proven to be a powerful tool to analyze large biological data. It helps to identify common features as well as distinctions among different diseases, disease subtypes, or among different biological processes. The resulted frequent clusters may provide new insights on the pathway/function networks. In the case of disease study, the results lead to new directions for biomarker and drug target discovery. The advantages of this workflow include the highly efficient processing of large biological data generated from high-throughput experiments, quick identification of highly correlated interaction networks, substantial reduction of the data dimensionality to a manageable number of variables for downstream comparative analysis, and consequently increased statistical power for detecting differences between conditions.

Molecular and Biological Characterization of Transient Antithrombin Deficiency: A New Concept in Congenital Thrombophilia

Antithrombin deficiency, mainly but not exclusively due to SERPINC1 gene variants, is a major thrombophilia that is significantly associated to early-onset venous thromboembolism. The diagnostic algorithm of antithrombin deficiency relies on the classical biochemical-molecular sequence, adopted for all thrombophilic states. Therefore, genetic analysis of SERPINC1 is restricted to cases with confirmed antithrombin deficiency, that is, cases with at least two positive findings by using functional assays or with other relatives carrying this disorder. This strategy has enabled the identification of gene variants in up to 80% of cases with antithrombin deficiency and rendered plenty of both biochemical and genetic knowledge about antithrombin. Furthermore, defects of N-glycosylation underlie a proportion of cases with antithrombin deficiency that is not explained by SERPINC1 variants.Nevertheless, diagnosis of antithrombin deficiency still encloses some uncertain puzzling questions. Although the prevalence of antithrombin deficiency is tremendously low, the use of high-throughput nucleotide sequencing for genetic analysis of SERPINC1 in consecutive patients with thromboembolic events suggests that antithrombin deficiency might be and underestimated disorder that surreptitiously increases thrombotic risk. Additionally, false negative results by using functional methods for antithrombin deficiency screening have been reported, all involving type II deficiencies.The present study aimed to identify gene defects and mechanisms involved in a specific type of antithrombin deficiency that might be elusive to an easy diagnosis to the classic diagnostic strategy. We addressed this aim with an original approach, the selection of cases with at least a positive finding by functional methods that however was not confirmed by a second analysis in other laboratory or in other sample from the same patient: what we have called transient antithrombin deficiency.This work included a total of 444 consecutive unrelated subjects, referred to our centre from more than 20 European hospitals during 23 years (1998-2021), with potential antithrombin deficiency, based on at least one positive functional assay performed at the hospital of origin. At least a new sample from all patients was delivered to our centre, so a new functional assay (a uniform anti-FXa assay for all recruited samples) was carried out for validation.By performing a full clinical, biological and characterization, a genetic defect was observed in 84.6% of 305 cases with constitutive deficiency, those with consistent deficiency in all tested samples: 248 in SERPINC1 and 10 had N-glycosylation defects. These results are fully compatible with those obtained from other large cohorts, supporting the high success rate of identification of a SERPINC1 genetic variant in these patients, and confirmed the relevance of disorders of glycosylation among cases with not SERPINC1 defect. But more interestingly, our study identified a molecular basis explaining antithrombin deficiency in 43.9% of 139 cases who had normal antithrombin activity in at least one sample, what we called transient antithrombin deficiency. These 61 cases, all with thrombosis, had missense SERPINC1 mutations (N=48), with two recurrent mutations (p.Ala416Ser, antithrombin Cambridge II, N=15 and p.Val30Glu, antithrombin Dublin, N=12) or N-glycosylation defects (N=13). Two mechanisms explained transient deficiency: the limitation of current functional methods to detect some variants, and the influence of external factors (conformational stress, generation of thrombin, alcohol intake) on the pathogenic consequences of these mutations.Our study supports that antithrombin deficiency is underestimated and cases with moderate risk of thrombosis may be missed if only functional methods and classical diagnostic algorithms are used. Furthermore, although we must take into consideration that most cases with transient deficiency are explained by acquired deficiency and laboratory mistakes, this work shows new evidences supporting that the pathogenic effect of a gene defect may be modulated by external factors, changing the paradigm of congenital thrombophilia and making it also a transient disorder. DisclosuresNo relevant conflicts of interest to declare.

Next-generation sequencing for the management of sarcomas with no known driver mutations.

Next-generation sequencing (NGS) has enabled fast, high-throughput nucleotide sequencing and has begun to be implemented into clinical practice for genomic-guided precision medicine in various cancer types. This review will discuss recent evidence that highlights opportunities for NGS to improve outcomes in sarcomas that have complex genomic profiles with no known driver mutations. Global genomic signatures detectable by NGS including tumour mutational burden and microsatellite instability have potential as biomarkers for response to immunotherapy in certain sarcoma subtypes including angiosarcomas. Identification of hallmarks associated with 'BRCAness' and homologous recombination repair defects in leiomyosarcomas and osteosarcomas may predict sensitivity to poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitors. Lastly, the use of NGS for evaluating cancer predisposition in sarcomas may be useful for early detection, screening and surveillance. Currently, the implementation of NGS for every sarcoma patient is not practical or useful. However, adopting NGS as a complementary approach in sarcomas with complex genomics and those with limited treatment options has the potential to deliver precision medicine to a subgroup of patients, with novel therapies such as immune checkpoint and PARP inhibitors. Moving forward, molecular tumour boards incorporating multidisciplinary teams of pathologists, oncologists and genomic specialists to interpret NGS data will complement existing tools in diagnosis and treatment decision making in sarcoma patients.

Correlation Analysis Among Genotype Resistance, Phenotype Resistance and Eradication Effect of Helicobacter pylori

BackgroundIt has not been fully confirmed whether the detection of Helicobacter pylori resistance gene mutation can replace antibiotic drug sensitivity test to guide the clinical individualized treatment. Therefore, we have studied this aspect and discussed the application value of antibiotic sensitivity gene test.Materials and MethodsThe biopsy specimen of gastric mucosa from the patients examined by endoscopy and positive for rapid urease test were collected continuously for histopathological analysis, H. pylori culture, antibiotic drug sensitivity test (E-test drug sensitivity test), and antibiotic sensitivity gene test (high-throughput nucleotide sequencing). The participants received triple plus bismuth solution eradication treatment (esomeprazole 20 mg, amoxicillin 1000 mg, clarithromycin 500 mg, and bismuth potassium citrate 220 mg, twice daily for 14 days) for follow-up, and the eradication effect was determined.ResultsThe 551/602 subjects, who met the inclusion criteria, were subjected to culture for H. pylori and antibiotic drug sensitivity determination; among them 506 were cultured successfully. The results showed that the resistance rates of H. pylori were 38.9% for clarithromycin and 31.0% for levofloxacin. In 489 H. pylori strains, the mutations were detected in clarithromycin and levofloxacin resistance genes, indicating the genotype resistance. The resistance genes of clarithromycin and levofloxacin were consistent with phenotype resistance with respect to sensitivity (81.2% and 69.7% for clarithromycin and levofloxacin, respectively) and specificity (88.9% and 93.7% for clarithromycin and levofloxacin, respectively). The eradication rate of H. pylori in the clarithromycin-resistant group was significantly lower than that in the sensitive group (ITT: 52.1% vs 85.0%, P < 0.001).ConclusionA correlation was established between the resistance genes of clarithromycin and levofloxacin and their phenotypic resistance and clinical efficacy. The detection of H. pylori resistance genes has a good clinical application prospect.

Novel Approach to Overcome Defects of Cell-SELEX in Developing Aptamers against Aspartate β-Hydroxylase.

Cell-based aptamer selection (Cell-SELEX) against predefined protein targets that benefits using the native form of the targets is the most promising approach to achieve aptamer probes capable of recognizing targets under both in vitro and in vivo conditions. The major disadvantages in Cell-SELEX are the imperfectness of the negative selection step and the lengthy procedure of selection. Here, we introduced the Counter-SELEX as part of our modified Cell-SELEX and implemented deep sequencing to overcome these shortcomings in developing aptamers against aspartate β-hydroxylase (ASPH) as a known tumor marker. In parallel with the conventional Cell-SELEX, five consecutive cycles of counter selection were accomplished using sequences bound to negative cells (the Counter-SELEX) to detect oligos that are not specific for ASPH. After high-throughput sequencing, the representative of each promising achieved family was subjected to further confirmatory analysis via flow cytometry, followed by the fluorescence immunostaining of histopathological sections. Implementing our innovative complementary method, annoying mis-selected sequences in Cell-SELEX enriched pools were effectively identified and removed. According to the affinity assay on the cells displaying ASPH, three aptamers, AP-Cell 1, AP-Cell 2, and AP-Cell 3, with Kd values of 47.51, 39.38, and 65.23 nM, respectively, were obtained, while AP-Cell 1 and 3 could then successfully spot ASPH displayed on the tissues. Our study showed that the Counter-SELEX could be considered as a complementary method for Cell-SELEX to overcome the imperfectness of the negative selection step. Moreover, high-throughput nucleotide sequencing could help to shorten the overall process.

Current status of use of high throughput nucleotide sequencing in rheumatology

ObjectiveHere, we assess the usage of high throughput sequencing (HTS) in rheumatic research and the availability of public HTS data of rheumatic samples.MethodsWe performed a semiautomated literature review on PubMed,...

Precision Medicine Based on High-Throughput Nucleotide Sequencing for a Patient with SCN2A-Related Developmental Epileptic Encephalopathy

Precision Medicine Based on High-Throughput Nucleotide Sequencing for a Patient with SCN2A-Related Developmental Epileptic Encephalopathy

Can Next-generation Sequencing Replace Fecal Immunochemical Tests or CT in the Screening of Colorectal Cancer and Advanced Adenoma?

To explore the feasibility of next-generation sequencing (NGS) for the screening of colorectal cancer (CRC) and advanced adenoma (AA). Observational study. Place and Duration of the Study: Renmin Hospital of Wuhan University, Wuhan, China, from June 2019 to February 2020. Patients who met inclusion and exclusion criteria were divided into three groups: CRC, AA, and control group. Then, the gene methylation status in the blood in the CRC, AA, and control group was analysed by NGS, and the CRC screening risk assessment model was used for comprehensive analysis. Afterwards, the methylated haplotype index (PHF Index) was calculated, and the screening results of the patients were classified as positive or negative, according to the score. The clinicopathological results were used as the gold standard, and the screening results for NGS were compared with the computed tomography (CT) and fecal immunochemical test (FIT) to text its feasibility. NGS has a certain detection ability for CRC, with a sensitivity of 57.1% (8/14). This was higher than that of FIT and CT, and the combined positive rate of these three methods could reach 92.3% (12/13). The sensitivity of detection of AA could reach up to 75.0% (6/8) after combining with FIT and CT. The positive rate of the NGS test for postoperative CRC was 23.1% (3/13), which was significantly lower than preoperative CRC. The sensitivity of CT for preoperative CRC detection was only 45.5% (5/11), but the specificity could reach up to 98.2% (55/56), which was higher than NGS (71/78, 91.0%) and FIT (27/33, 81.8%). Although NGS cannot replace FIT and CT at present, this provides a new effective and auxiliary detection method for people who are unsuitable or unwilling to receive colonoscopy. Key Words: Colorectal neoplasms, Adenoma, High-throughput nucleotide sequencing, Mass screening, Fecal immunochemical tests, Computed tomography.

High-throughput nucleotide sequencing for bacteriome studies in oral squamous cell carcinoma: a systematic review.

Dysbiosis has been identified in oral squamous cell carcinoma (OSCC). The aim of this study was to carry out a systematic review of an electronic research that was carried out on articles published between January 2008 and September 2018. Eight studies were selected after applying the inclusion and exclusion criteria. All articles targeted the hypervariable regions of the 16S rRNA gene. At the phylum level, it was found reduction of Bacteroidetes (2/8 studies) and increase of Firmicutes (2/8 studies). At the genus level, Rothia increased (1/8 studies) and decreased (2/8 studies) in tumor samples, and Streptococcus also was found increased (3/8 studies) and reduced (3/8 studies). Fusobacterium only increased in OSCC samples (3/8 studies). At species level, an increase in F. nucleatum subsp. polymorphum was more associated to OSCC (2/8 studies) than with controls, as was P. aeruginosa (3/8 studies). In summary, the results corroborated dysbiosis in OSCC patients, with enrichment of microbial taxa that are associated with inflammation and production of acetaldehyde. However, variations of study design and sample size were observed among the studies, as well as a shortage of more detailed analyses of possible correlations between risk habits and OSCC. This lack of more detailed analysis may be the cause of the inconsistencies in regard of the alterations reported for certain genera and species. In conclusion, there is an association between OSCC and oral microbiota dysbiosis, but its role in oral carcinogenesis needs to be clarified in more detail.

Enhancers, development, and evolution.

A single-celled fertilized egg develops into a complex, multicellular animal through a series of selection processes of developmental pathways. During these processes, regulatory genes exhibit spatiotemporally restricted expression under the control of the species-specific genetic program, and dictate developmental processes from germ layer formation to cellular differentiation. Elucidation of molecular mechanisms underlying developmental processes and also of mechanistic bases for morphological diversification during evolution is one of the central issues in contemporary developmental biology. Progress has been made due to recent technological innovations, such as high-throughput nucleotide sequencing, live-cell imaging, efficient genetic manipulation, and establishment of the organoid system, opening new avenues to the above issues.

Analysis of gut microbiota change in colorectal adenomatous polyps patients by 16S rRNA sequencing

Objective To explore the characteristics of gut microbiota change in colorectal adenomatous polyps (CAP), which has been considered as precancerous lesion for colorectal cancer. Methods Thirty patients with colon adenomatous polyps (CAP group) and thirty healthy individuals without adenomatous polyps (HC group) who underwent colonoscopy at the First Affiliated Hospital of Kunming Medical University from November 2017 to April 2018 were randomly collected. The biopsy mucosae were collected by endoscopic electrocoagulation, and DNA was extracted to amplify 16S rRNA V3-V4 region, followed high-throughput sequencing with Illumina MiSeq platform. The experimental results were analyzed using Wilcoxon test. Results The alpha diversity of CAP patients was higher than that of healthy controls (Chao & Ace P<0.01). A decreased abundance of Bacteroidetes (FC=0.38) was observed at phylum level(P<0.05). At genus level, the abundances of Bacteroides (FC=0.32) , Escherichia (FC=0.57) , Ruminococcus (FC=0.42) , Blautia (FC=0.27) , and Dorea (FC=0.57) were decreased (P<0.05), but those of Pseudomonas(FC=2.43), Lactococcus(FC=2.84), Geobacillus(FC=2.07), and Acinetibacter(FC=2.36) were increased in CAP patients (P<0.05). Conclusions Compared with healthy volunteers, there are significant differences in the abundance and diversity of the adenoma tissue in CAP patients, indicating that there is an imbalance of gut microbiota in the adenomatous polyps. The imbalance of intestinal microenvironment may contribute to the occurrence and development of CAP. Key words: Colonic neoplasms; Adenomatous polyps; Gastrointestinal microbiome; RNA, ribosomal, 16S; High-throughput nucleotide sequencing

Characteristics of gene mutations in Chinese single-center pulmonary adenocarcinoma patients and comparison with European and American pulmonary adenocarcinoma populations

Objective To explore the characteristics and significances of gene mutations in pulmonary adenocarcinoma, and to provide evidence for targeted medication. Methods High throughput sequencing based target-capture sequencing was performed in 104 patients with pulmonary adenocarcinoma to detect the mutational status of 56 cancer-related genes. All patients were diagnosed in the First People's Hospital of Kunshan from May 2017 to August 2018. The mutational characteristics of pulmonary adenocarcinoma was analyzed and compared with European and American pulmonary adenocarcinoma populations. The correlations between mutational characteristics and clinical features were analyzed, and the mutation sites for targeted medication were screened. Results Among 104 patients with pulmonary adenocarcinoma, totally 34 mutational genes were detected in 84 patients (81%, 84/104). Highly frequent mutations included epidermal growth factor receptor (EGFR) (49%, 51/104), TP53 (21%, 22/104), KRAS (13%, 14/104), and BRAF (6%, 6/104). Among all the 187 variants, 76% (142/187) were non-synonymous missense mutations, 13% (24/187) were small fragment deletions, 6% (12/187) were copy number variants, 3% (5/187) were small fragment insertions, and 2% (4/187) were nonsense site mutations. Among 104 patients with pulmonary adenocarcinoma, 34 targeted drug-associated mutations of 13 genes were detected in 68 patients (65%), and 19 (18%) patients harbored ≥ 2 targeted drug-associated mutations. EGFR mutations were more common in female patients than in male patients [62% (34/55)vs. 35% (17/49), χ2= 7.629, P= 0.006], while KRAS mutations were more frequent in male patients than in female patients [22% (11/49) vs. 5% (3/55), χ2= 6.424, P= 0.011]. The mutation frequencies of gene EGFR, TP53, KRAS, and CDKN2A in Chinese single-center (the First People's Hospital of Kunshan) and European and American adenocarcinoma populations were significantly different (all P < 0.05). Conclusions The molecular mutational characteristics of pulmonary adenocarcinoma are complex, and vary greatly among different populations. High throughput sequencing-based multiple-gene detection can reveal its mutational features comprehensively, and that has important roles in personal targeted medication guidance, drug-resistance monitoring and prognosis evaluation. Key words: Lung neoplasms; Adenocarcinoma; High-throughput nucleotide sequencing; Mutation; Molecular targeted therapy

Progress of big data analysis and artificial intelligence technology for hematologic neoplasms

The new wave of artificial intelligence pushed by deep learning algorithms has dramatically promoted the development of big data analysis technology. On the other hand, advances in life sciences represented by high-throughput genome sequencing have provided massive medical data. Artificial intelligence technology has also provided a powerful tool for hematological malignancy research. This article introduces related research progress in the 61st American Society of Hematology Annual Meeting. Key words: Hematologic neoplasms; Artificial intelligence; High-throughput nucleotide sequencing; Big data

Application of metagenomics next-generation sequencing in monitoring Legionella pneumophila infection after allogeneic hematopoietic stem cell transplantation

Objective To investigate the application of metagenomic next-generation sequencing (mNGS) in detection of the rare or difficult-to-cultivate pathogens. Methods One patient with acute lymphoblastic leukemia who went through allogeneic hematopoietic stem cell transplantation (allo-HSCT) developed symptoms of infection after transplantation. Conventional microbial culture, polymerase chain reaction (PCR), and mNGS combined with biological information analysis were performed with plasma and cerebrospinal fluid samples, the anti-infective treatment was adjusted according to the test results, and the efficacy was assessed. Results No suspected pathogens were detected by microbial culture and PCR in the cerebrospinal fluid and plasma samples since the patient developed infection symptoms. However, Legionella pneumophila was analyzed by mNGS in the cerebrospinal fluid specimen on day 23 after allo-HSCT (reads count: 19 655), and it was considered as the principal pathogen after comprehensively evaluating the patient's clinical manifestations and the test results. Then the antimicrobial treatments were adjusted according to the patient's clinical manifestations and laboratory test results, and the number of gene sequences of Legionella pneumophila was monitored by mNGS method. Azithromycin, tigecycline, and other antibiotics effective for Legionella pneumophila were used after detecting this pathogen. A total of 15 mNGS analysis were performed during the 5-month period, and the highest number of Legionella pneumophila sequences monitored in the cerebrospinal fluid was 2 226, the lowest was 253 and eventually turned negative. The clinical symptoms and treatment outcomes were consistent with the mNGS monitoring results. Conclusions The mNGS technology has significant value in detection of the rare and difficult-to-cultivate pathogens. The mNGS technology provides a valuable supplement to microbial culture and PCR methods. Key words: High-throughput nucleotide sequencing; Metagenome; Hematopoietic stem cell transplantation; Legionella pneumophila

Application progress of transcriptome sequencing in hematological malignancies

Transcriptome sequencing (RNA-seq) has unique advantages in analyzing gene fusion, splicing mutations, and gene expression profiles. Single-cell RNA-seq provides powerful tools to reveal cellular heterogeneity in normal and tumor tissues. With the widespread application of high-throughput gene sequencing technology and the rapid reduction in cost, RNA-seq is increasingly used in hematological malignancies research. This article introduces the related research progress in conjunction with reports at the 61st American Society of Hematology Annual Meeting. Key words: High-throughput nucleotide sequencing; Transcriptome; Gene sequencing; Fusion genes; Gene mutations; Hematological malignancies

Application of cerebrospinal fluid circulating cell-free DNA in the diagnosis and treatment of leptomeningeal metastases from non-small-cell lung cancer

Objective To investigate the application value of cerebrospinal fluid circulating cell-free DNA (cfDNA) in the diagnosis and treatment of leptomeningeal metastases in non-small-cell lung cancer (NSCLC). Methods Twenty-five patients with leptomeningeal metastases of NCSLC from Fudan University Huashan Hospital North during the period from September 2017 to November 2018 were enrolled. All 25 patients were confirmed leptomeningeal metastases by cerebrospinal fluid cytology and immunocytochemical staining of cytokeratin(CK7), carcinoembryonic antigen(CEA), thyroid transcription factor-1(TTF-1) and Ki67. The cerebrospinal fluid cfDNA was extracted and genetic variation of 12 genes including epidermal growth factor receptor(EGFR), TP53 and anaplastic lymphoma kinase(ALK) was detected by next-generation sequencing [PlasAim TM gene non-invasive detection of lung cancer (12 gene) kit, Singlera Genomics].The application value of cerebrospinal fluid cfDNA in the diagnosis and treatment of leptomeningeal metastases of NSCLC was analyzed with the cfDNA mutation data and the clinical follow-ups. Results Morphologically typical lung cancer tumor cells with tumor immunochemistry markerCK, CK7 and CEA were found in the cerebrospinal fluid of all 25 patients. Next generation sequencing of cerebrospinal fluid showed that 96% (24/25) patients had at least one single nucleotide variation (SNV) or copy number variation (CNV). The EGFR and TP53 mutations were identified in 80% (20/25) and 48%(12/25) of the patients, respectively. In addition, patients with bone metastases had a higher rate of EGFR mutations than those without bone metastases (100% vs 64%, P<0.05). Changes in the mutant allele frequency of EGFR and TP53 in cerebrospinal fluid were consistent with patients′ disease progression parameters including neurological symptoms, imaging, and tumor biomarkers. The results indicate that genetic alteration of EGFR in cerebrospinal fluid cfDNA is an actionable biomarker for targeted therapy of leptomeningeal metastases of lung cancer. Conclusion Cerebrospinal fluid cfDNA accurately reveals the unique genetic background of leptomeningeal metastasis in NSCLC, showing great application value in the diagnosis and treatment of the leptomeningeal metastasis of NSCLC. Key words: Carcinoma, non-small-cell lung; Neoplasm metastasis; Meningeal neoplasms; Cell-free nucleic acids; Cerebrospinal fluid; High-throughput nucleotide sequencing